Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, 145 Xingda Road, Taichung 402, Taiwan.
Institute of Molecular Biology, College of Life Science, National Chung Hsing University, 145 Xingda Road, Taichung 402, Taiwan; The iEGG and Animal Biotechnology Center, National Chung Hsing University, 250 Kuo Kuang Road, Taichung 40227, Taiwan.
Gene. 2023 May 30;867:147371. doi: 10.1016/j.gene.2023.147371. Epub 2023 Mar 16.
Beak and feather disease virus (BFDV) belongs to the Circoviridae family, which has a relatively simple replication mechanism. As BFDV lacks a mature cell culture system, a novel mini-replicon system based on the reporter plasmid that contains the origin of replication, which can bind to the Rep protein expressed from another plasmid and thus trigger its replication and induce/increase luminescence was developed. The dual-luciferase assay was used in this system to measure replicative efficiency by comparing relative light units (RLU) of firefly luciferase. Linear relationships between the luciferase activity of the reporter plasmids with the BFDV origin of replication and the amounts of the Rep protein and vice versa were found, suggesting the mini-replicon system can be used to quantify viral replication. Moreover, the activities of reporter plasmids driven by mutated Rep proteins or the activities of reporter plasmids with mutations were significantly downregulated. The Rep and Cap promoter activities can be characterized using this luciferase reporter system. Notably, the RLU of the reporter plasmid was considerably inhibited in the presence of sodium orthovanadate (NaVO). When BFDV-infected birds were treated with NaVO, the viral loads of BFDV rapidly decreased. In conclusion, this mini-replicon reporter gene-based system provides a practical means to screen for anti-viral drug candidates.
喙羽病病毒（BFDV）属于圆环病毒科，其复制机制相对简单。由于 BFDV 缺乏成熟的细胞培养系统，因此开发了一种基于报告质粒的新型微小复制子系统，该系统包含复制起点，可与来自另一个质粒的 Rep 蛋白结合，从而触发其复制并诱导/增加发光。该系统使用双荧光素酶测定法通过比较萤火虫荧光素酶的相对发光单位（RLU）来测量复制效率。发现报告质粒的荧光素酶活性与 BFDV 复制起点的数量以及 Rep 蛋白之间存在线性关系，反之亦然，这表明微小复制子系统可用于定量病毒复制。此外，突变的 Rep 蛋白驱动的报告质粒的活性或具有突变的报告质粒的活性均显著下调。可以使用此荧光素酶报告系统来表征 Rep 和 Cap 启动子的活性。值得注意的是，在存在偏钒酸钠（NaVO）的情况下，报告质粒的 RLU 受到明显抑制。当用 NaVO 处理感染 BFDV 的鸟类时，BFDV 的病毒载量迅速下降。总之，基于该微小复制子报告基因的系统为筛选抗病毒药物候选物提供了一种实用手段。
